I have read the journal's policy and the authors of this manuscript have the following competing interests: EMD and CS have received research funding from Pfizer for an unrelated project. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

* E-mail: fmruss@unimelb.edu.au2910201920191410e02243921472019131020192019Dunne et alThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Nasopharyngeal carriage of Streptococcus pneumoniae (the pneumococcus) is a precursor to pneumococcal disease. Several host and environmental factors have been associated with pneumococcal carriage, however few studies have examined the relationship between host factors and pneumococcal carriage density. We sought to identify risk factors for pneumococcal carriage and density using data from cross-sectional pneumococcal carriage surveys conducted in the Lao People's Democratic Republic before and after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13). Nasopharyngeal swabs were collected infants from aged 5–8 weeks old (n = 999) and children aged 12–23 months (n = 1,010), pneumococci detected by quantitative PCR, and a risk factor questionnaire completed. Logistic and linear regression models were used to evaluate associations between participant characteristics and pneumococcal carriage and density. In infants aged 5–8 weeks, living in a household with two or more children under the age of five years (aOR 1.97; 95% CI 1.39–2.79) and low family income (aOR 1.64; 95% CI 0.99–2.72) were positively associated with pneumococcal carriage. For children aged 12–23 months, upper respiratory tract infection (URTI) symptoms (aOR 2.64; 95% CI 1.97–3.53), two or more children under five in the household (aOR 2.40; 95% CI 1.80–3.20), and rural residence (aOR 1.84, 95% CI 1.35–2.50) were positively associated with pneumococcal carriage. PCV13 vaccination was negatively associated with carriage of PCV13 serotypes (aOR 0.60; 95% CI 0.44–0.83). URTI symptoms (p < 0.001), current breastfeeding (p = 0.005), rural residence (p = 0.012), and delivery by Caesarean section (p = 0.035) were associated with higher mean pneumococcal density in pneumococcal carriers (both age groups combined). This study provides new data on pneumococcal carriage and density in a high disease burden setting in southeast Asia.

http://dx.doi.org/10.13039/100001125GAVI AllianceRussellFiona M.http://dx.doi.org/10.13039/501100000925National Health and Medical Research CouncilThe project was funded by Gavi, the Vaccine Alliance (https://www.gavi.org/) and the World Health Organization Western Pacific Regional Office (https://www.who.int/westernpacific), with support from the Victorian Government’s Operational Infrastructure Support Program (https://www2.health.vic.gov.au/about/clinical-trials-and-research/operational-infrastructure-support). FMR was supported by a NHMRC Early Career and TRIP Fellowships (https://www.nhmrc.gov.au/). CS was supported by a NHMRC Career Development Fellowship (1087957) and a veski Inspiring Women Fellowship (https://www.veski.org.au/). EFGN holds an Australian Government Research Training Scholarship (https://www.education.gov.au/research-training-program). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Data AvailabilityAll relevant data are not publicly available because the ethics committee approved the study protocol, which specified the study objectives and how data would be used. These restrictions apply to all data included in this manuscript. It is not consistent with our ethical permissions to share de-identified or aggregate versions of our data, as publicly available data could be used for purposes that were not specified in the protocol approved by the ethics committee, and therefore would be a breach of our ethics permissions. During the informed consent, the purpose of the study was explained to participants, and they were told how their data would be used. The use of these data for a new purpose that was not included in the approved study protocol would require additional ethical approval from the Lao PDR Ministry of Health National Ethics Committee for Health Research. Following approval, de-identified data would be made available. Additionally, this process is mindful of potential sensitivities regarding data from ethnic minorities. We have included contact information for the ethics committee via Dr Sengchanh Kounnavong, sengchanhkhounnavong@hotmail.com. We recommend that requests for data also be sent to the Principal Investigator, Prof. Fiona Russell (fmruss@unimelb.edu.au), so she can assist with the process.Introduction

The bacterium Streptococcus pneumoniae (the pneumococcus) is a significant global pathogen, and in 2015 was responsible for an estimated 317,300 deaths, most due to pneumonia, in children under five years old.[1] The majority of pneumococcal disease burden occurs in low- and middle- income countries (LMICs), believed to be due to poor access to health care and pneumococcal vaccines, and higher rates of risk factors such as HIV infection and malnutrition.[2] Other risk factors such as overcrowding contribute to the higher burden of pneumococcal disease in LMICs.[3]

Pneumococci are commonly carried in the nasopharynx of young children, with reported prevalence rates in LMICs ranging from 6 to 93%.[3] Although pneumococcal carriage is typically asymptomatic, it is a precursor for the development of pneumococcal disease and serves as the reservoir for this exclusively human pathogen.[4] High pneumococcal density (bacterial load) in the nasopharynx is associated with lower respiratory tract infections and pediatric pneumonia.[5]. In mouse models, high pneumococcal density in the nasopharynx increases transmission.[6]

Several host, socio-economic, and environmental factors are risk factors for pneumococcal carriage. These include day care attendance, living in a household with other young children, low socio-economic status, and symptoms of an upper respiratory tract infection (URTI).[7–11] In some studies, children in rural areas had higher odds of pneumococcal carriage compared to those living in urban settings.[12, 13] Co-infection with respiratory viruses, URTI symptoms, and low family income have been linked to higher pneumococcal density in children.[5, 9, 14, 15]

The Lao People’s Democratic Republic (Lao PDR) is a lower-middle income country in East Asia. Lao PDR has a mortality rate of 67 per 1000 children under five, and a high burden of pneumonia in children. (https://www.gavi.org/country/lao-pdr/ [Accessed 13 June 2019]). The 13-valent pneumococcal conjugate vaccine (PCV13), which provides protection against 13 pneumococcal serotypes, was introduced into the national infant immunization schedule in Lao PDR in late 2013. Previously, we conducted nasopharyngeal carriage surveys before and two years after PCV13 introduction to evaluate the impact of the PCV program on pneumococcal carriage in two age groups: young children aged 12–23 months (the majority of whom were PCV-vaccinated in the post-PCV13 survey), and unvaccinated infants aged 5–8 weeks.[16] Following PCV13 introduction, the carriage prevalence of overall pneumococci did not change. Prevalence of PCV13-serotypes declined significantly in 12–23 month old children (from 33% to 20%; adjusted prevalence ratio 0.77 [95% CI 0.61–0.96], and there was some evidence of indirect effects in 5–8 week old infants (decline from 7% to 5%, adjusted prevalence ratio 0.74 [95% CI 0.43–1.27). Here, we describe a secondary analysis conducted using data from these cross-sectional surveys to identify demographic and household factors associated with carriage of pneumococci overall, PCV13 serotype carriage, and pneumococcal carriage density.

Materials and methodsStudy design and participants

Cross-sectional nasopharyngeal carriage surveys were conducted from November 2013—February 2014 (“pre-PCV”), and November 2015—February 2016 (“post-PCV”) as described previously.[16] Participants were enrolled in Vientiane, the capital of Lao PDR, and the rural Bolikhamxay Province. Urban participants were enrolled from maternal and child health centers during routine clinic visits. Rural participants were enrolled during maternal and child health visits and visits to surrounding households. Inclusion criteria were age (5–8 week old infants and 12–23 month old children), temperature ≤ 37°C per axilla, and having lived in the area for at least three consecutive months. Previous receipt of PCV13 was an exclusion criteria for children and infants during the first survey, for infants aged 5–8 weeks for the second survey. Nasopharyngeal swabs were collected and stored according to WHO guidelines.[17] Study staff completed a questionnaire to collect data on demographics and potential risk factors (chosen based upon the literature) for each participant. These data included: sex, ethnicity (self-reported), urban or rural residence, the presence of URTI symptoms (including coryza, allergic rhinitis, or cough), antibiotic usage in the previous two weeks, exposure to household cigarette smoke, main source of cooking fuel (wood, charcoal, gas, electricity, kerosene), number and ages of other children in the household, family income (Kip/week), mode of delivery, and current breastfeeding status. A binary value for poverty was created using the $1.25 USD per day value international poverty line recommended by the World Bank from 2008–2015 (http://www.worldbank.org/). PCV13 immunization history was obtained from participant vaccination status cards or health center records.

The study was undertaken according to the protocol approved by the Lao PDR Ministry of Health National Ethics Committee for Health Research (061-NECHR), the Western Pacific Regional Office Ethics Review Committee, and The Royal Children’s Hospital/Murdoch Children’s Research Institute Human Research Ethics Committee (HREC 33177A/HREC 33177B). Written informed consent was obtained from the parent or guardian for all participants.

Laboratory procedures

Samples were stored in ultra-low temperature freezers at the Lao-Oxford-Mahosot Hospital Wellcome Trust Research Unit (LOMWRU, Vientiane, Lao PDR) until shipment on dry ice to the Murdoch Children’s Research Institute (Parkville, Australia) for microbiological analysis. Detection and quantification of pneumococci and pneumococcal serotyping were performed as previously described.[16] In brief, DNA extraction of 100 μl swab media was conducted using a MagNA Pure LC instrument (Roche) and pneumococci were detected and quantified by real-time quantitative PCR (qPCR) targeting the lytA gene.[18] A standard curve prepared using genomic DNA extracted from a reference isolate of S. pneumoniae (ATCC 6305) was used for quantification. The standard curve also served as a positive control, and extraction controls and no template controls were included as negative controls in each qPCR run.

Pneumococcal density data were reported in genome equivalents/ml (GE/ml). This unit approximates pneumococcal density with the assumption that each pneumococcal cell contains one genome (with a genome size of 2 Mb) and each genome contains one copy of the lytA gene, and an extraction volume of 0.1 ml swab media.

Pneumococcal-positive samples underwent molecular serotyping by microarray following a culture-amplification step on horse blood agar plates containing 5 μg/ml of gentamicin (Oxoid). DNA was extracted using a QIAcube HT instrument (Qiagen) and microarray performed using the Senti-SPv1.5 microarray (BUGS Bioscience).[19]

Statistical analysis

Data entry and cleaning were conducted using EpiData version 3.1 and Stata version 15.1 as previously described.[16] Statistical analyses was performed using Stata 15.0. To evaluate the relationship between participant characteristics and pneumococcal carriage, unadjusted and adjusted odds ratios and 95% confidence intervals (CIs) were determined using logistic regression. Potential risk factors were examined separately for each age group for overall pneumococcal carriage. The following variables (shown in Table 1) were evaluated in the univariable analysis: sex, location (urban or rural), ethnicity, the presence of upper URTI symptoms, antibiotic use in the previous two weeks, exposure to cigarette smoke, primary source of fuel, two or more children under the age of five years in the household, family income below the poverty line, mode of delivery, breastfeeding status, and survey year. Primary source of fuel was categorized into biofuels (wood and charcoal) or non-biofuels (electricity, gas, and kerosene) and ethnicity data were reclassified as Lao Loum or other prior to analysis. For 12–23 month old children, PCV13 vaccination status was also assessed, however survey year was omitted due to co-linearity with PCV13 status. Adjusted odds ratios and 95% CI were calculated using multivariable logistic regression models included variables selected a priori based on the literature (URTI symptoms, two or more children under the age of five years in the household, poverty) and any variables with p < 0.2 by univariable analysis. Similar analyses were also undertaken for PCV13 serotype carriage for the 12–23 month old age group. When serotyping results were not available from a sample due to technical issues, that sample was excluded from serotype-specific analyses.

10.1371/journal.pone.0224392.t001Table 1

Characteristics of pneumococcal carriage survey participants in Lao PDR, conducted before and two years after PCV13 introduction.

Characteristics

5–8 week old infants(n = 999)a

12–23 month old children(n = 1,010)a

Survey, n (%)

Pre-PCV13

498 (49.9)

503 (49.8)

Two years post-PCV13

501 (50.2)

507 (50.2)

Age. median (IQR)b

6.7 weeks(6.5–7.0)

16.6 months(15.6–20.0)

Male sex, n (%)

n = 998509 (51.0)

484 (47.9)

Ethnicity, n (%)

n = 998

Lao Loum

964 (96.6)

963 (95.4)

Lao Thung

3 (0.3)

13 (1.3)

Hmong

27 (2.7)

30 (3.0)

Other

4 (0.4)

4 (0.4)

Rural residence, n (%)

70 (7.0)

470 (46.5)

URTIcsymptoms, n (%)

162 (16.2)

625 (61.9)

Antibiotic use in the previous two weeks, n (%)

n = 99729 (2.9)

n = 1,007405 (40.2)

Exposure to household cigarette smoke, n (%)

n = 996366 (36.8)

n = 1,007440 (43.7)

Primary household fuel source, n (%)

n = 1,007

wood

164 (16.4)

227 (27.5)

charcoal

510 (51.1)

482 (47.8)

kerosene

1 (0.1)

1 (0.1)

gas

179 (19.7)

153 (15.2)

electricity

145 (15.4)

95 (9.4)

Two or more children < 5 years in the household, n (%)

n = 998409 (41.0)

349 (34.6)

Below poverty lined, n (%)

109 (10.6)

70 (6.9)

Delivered by Caesarean section, n (%)

238 (23.8)

179 (17.7)

Current breastfeeding, n (%)

n = 998844 (84.6)

n = 1,009201 (19.9)

PCV13 vaccinatede, n (%)

0 (0.0)

n = 1,000448 (44.8)

aIf participant data were missing for an individual variable, results for that variable were excluded and the denominator used was the number of participants with data available.

bIQR = interquartile range

cURTI = upper respiratory tract infection

dDefined as $1.25 USD/day

eReceived 2 or 3 doses of PCV13

Pneumococcal density data were log10 transformed prior to analysis. Potential relationships between potential risk factors described above and pneumococcal density we evaluated using linear regression. This analysis combined both age groups and was restricted to pneumococcal carriers. Multivariable linear regression models included URTI symptoms (selected a priori) plus variables with p < 0.2 by univariable analysis. Results were reported as the unadjusted and adjusted coefficients (the difference in mean density) with 95% CIs and p values.

Results

Table 1 summarizes characteristics of the 2,009 study participants. Generally, participant characteristics were similar between age groups with a few exceptions: very few of the 5–8 week old infants lived in rural areas, URTI symptoms and recent antibiotic use was much more common in 12–23 month old children, and breastfeeding was more common in the young infants. In the post-PCV survey, 448/497 (90.1%) of participants aged 12–23 months had received two or more doses of PCV13. Information on PCV13 immunization history was not available for ten participants.

One swab was excluded due to technical issues, and 9 swabs that were positive for pneumococcus were not able to be serotyped. For 5–8 week old infants, 157/999 (15.7%; 95% CI 13.5–18.1) carried pneumococcus, and 58/995 (5.8%; 95% CI 4.5–7.5) carried a PCV13 serotype. For the 12–23 month old children, 511/1009 (50.6%; 95% CI 47.5–53.8) carried pneumococcus, and 265/1005 (26.4%; 95% CI 23.7–29.2) carried a PCV13 serotype. Pneumococcal density was variable among carriers, ranging from 2.9 to 8.2 log10 GE/ml. The median carriage density was 5.8 log10 GE/ml (range 3.5–8.2; IQR 5.1–6.6) in 5–8 week old infants and 5.8 log10 GE/ml (range 2.9–8.1; IQR 5.0–6.5) in 12–23 month old children.

Associations between participant characteristics and overall pneumococcal carriage for each age group are shown in Tables 2 and 3. For 5–8 week old infants, living in a household with two or more children under the age of five years (aOR 1.97; 95% CI 1.39–2.79) was positively associated with pneumococcal carriage, and there was some evidence for a positive association with having a family income below the poverty line (aOR 1.64; 95% CI 0.99–2.71). Living in a household with two or more children under five was positively associated with pneumococcal carriage in children aged 12–23 months (aOR 2.40; 95% CI 1.80–3.20), along with rural residence (aOR 1.84; 95% CI 1.35–2.50) and having URTI symptoms (aOR 2.64; 95% CI 1.97–3.53). URTI symptoms were significantly more common in rural children (336/470, 71.5%) compared to children from urban areas (289/540, 53.7%, p < 0.001, chi-squared test).

Carriage of PCV13 serotypes was also examined, as these serotypes are more likely to cause invasive pneumococcal disease, and specifically targeted by the PCV immunization program. Due to the relatively small number of 5–8 week old infants who carried a PCV13 serotype, this analysis was only conducted for 12–23 month old children (Table 4). Factors identified to be positively associated with PCV13 serotype carriage were consistent with those associated with overall pneumococcal carriage (rural residence, presence of URTI symptoms, and living in a household with two or more children under five). Additionally, previous PCV13 vaccination was associated with reduced odds of PCV13 serotype carriage (aOR 0.60; 95% CI 0.44–0.83). To examine factors associated with PCV13 serotype carriage prior to PCV13 introduction, we conducted an analysis only including children from the pre-PCV survey. Results were consistent with those from the analysis of both surveys presented in Table 4: following multivariable analysis, rural living (aOR 1.70; 95% CI 1.08–2.68; p = 0.021), the presence of URTI symptoms (aOR 2.44; 95%CI 1.46–4.07, p = 0.001), and living in a household with two or more children under five (aOR 2.59; 95%CI 1.72–3.89; p < 0.001) were associated with PCV13 serotype carriage in the pre-PCV13 survey.

Southeast Asia has one of the highest estimated incidence of pneumococcal disease in children in the world.[1] However, relatively few studies have examined risk factors for pneumococcal carriage, a precursor to pneumococcal disease, in this setting. We examined carriage in infants aged 5–8 weeks old prior to their first dose of PCV13, as unvaccinated neonates and young infants are particularly vulnerable to invasive bacterial infections, including those caused by pneumococcus.[20] In this age group, having and two or more children under five in the household and low family income were risk factors for pneumococcal carriage. In Lao PDR children aged 12–23 months, URTI symptoms, two or more children under five years old in the household, and rural residence were associated with pneumococcal carriage. Results from our study were similar with other pneumococcal carriage studies conducted in the region. In Vietnam, age and day care attendance were associated with pneumococcal carriage in children under five.[21] In a longitudinal study of infants in Thailand followed from birth, maternal smoking and presence of other young children in the household were associated with earlier pneumococcal colonization.[22] Living in a household containing two or more children under five years old and URTI symptoms were identified as risk factors for pneumococcal carriage in Indonesian children aged 12–24 months.[9] Results from our study advance upon previous work by assessing risk factors for pneumococcal carriage in a unique population within which PCV has been recently introduced.

Lao PDR is a diverse nation, with 49 distinct ethnic groups, and historically, ethnic minority groups have had poorer health status, including increased childhood mortality and lower vaccination rates.[23] As our study was conducted in the capital city and a nearby province, relatively few participants belonging to ethnic minorities were enrolled, and therefore results are not representative of the overall population of Lao PDR. Nearly half of 12–23 month old participants, compared with 7% of 5–8 week old infants, lived in rural areas; this may explain why rural residence was identified as a risk factor only in the older age group. Consistent with a previous study on respiratory infection burden in Lao PDR, children in rural areas were more likely to have UTRI symptoms.[24]

Risk factors for PCV13 carriage were similar to those associated with overall pneumococcal carriage, and as expected, children who received two or three doses of PCV13 had reduced odds of carrying a PCV13 serotype. In later years following PCV13 introduction, carriage of PCV13 serotypes may become associated with contact with under-vaccinated communities and/or interactions with older, unvaccinated children and adolescents.[25]

In our study, pneumococcal carriage was not associated with recent antibiotic use; this may be due to the unreliability of parent-reported data on antibiotic use, which has been previously documented in Lao PDR, and high rates of antimicrobial resistance in pneumococci.[26] We previously reported that over 70% of pneumococcal-positive carriage samples harbored at least one antimicrobial resistance gene.[16] Results from studies examining cigarette exposure as a risk factor for pneumococcal carriage have reported varying results, and we did not find an association in our study population.[9, 27–29] However, exposure to cigarette smoke can be difficult to determine without monitoring, and levels of smoke exposure and maternal smoking were not assessed in our study. Indoor air pollution caused by the use of solid fuels such as wood and charcoal as cooking fuel is a risk factor for pneumonia in children.[30] In our study, biofuels were associated with increased pneumococcal carriage in 12–23 month old children by univariable analysis but not following multivariable analysis.

High pneumococcal density in the nasopharynx is associated with respiratory infections including pneumonia in children.[5, 31, 32] Additionally, pneumococcal density in the nasopharynx has been investigated as a potential diagnostic tool for pediatric pneumonia.[31] As such, it is important to understand what underlying host factors may influence pneumococcal carriage density, and our study provides new data on this topic. Consistent with other studies, individual pneumococcal carriage density varied widely.[33,34] We identified several factors associated with increased pneumococcal density. The presence of URTI symptoms was positively associated with pneumococcal density in our study, consistent with results from community carriage surveys in young children in Indonesia and Belgium.[9, 35] Living in a rural area was associated with increased pneumococcal density; this may be related to socio-economic and environmental differences between urban and rural households. Pneumococcal density was higher in the post-PCV carriage survey, but as this was the case for both PCV13 serotypes and non-PCV13 serotypes, this was not attributed to PCV.[16] We have performed pneumococcal density studies before and after PCV in Fiji, Mongolia, and Lao PDR. Similar to our findings in Lao PDR, density of both PCV13 and non-PCV13 serotypes was higher in children following PCV13 introduction in Mongolia.[36] In contrast, density of both vaccine serotypes and non-vaccine serotypes declined in children in Fiji following the introduction of PCV10.[34] In an experimental human pneumococcal challenge model, adults who received PCV13 had lower pneumococcal density compared with those who received a control vaccine.[37] To date, there is not consistent evidence of an effect of PCV on pneumococcal density in children. Variations in density observed in cross-sectional studies with carriage assessed during different years may be temporal and/or related to as number of unmeasured factors.

Two previously unreported factors were linked with higher pneumococcal density: delivery by Caesarean section and current breastfeeding. Unlike URTI symptoms and rural living, both of these factors were not associated with the likelihood of pneumococcal carriage, but rather with increased pneumococcal loads in pneumococcal carriers. Mode of delivery has been found to significantly affect the development of the nasopharyngeal microbiome through the first six months of life.[38] Caesarean birth was associated with a high abundance of Streptococcus from early life in a nasopharyngeal microbiome study of Dutch infants.[39] It is possible that Caesarean delivery may influence microbiome development in a manner that supports pneumococcal growth, however this finding requires further investigation. We did not collect detailed breastfeeding data on exclusive vs. non-exclusive breastfeeding, formula use, and duration of exclusive breastfeeding, so the relationship between breastfeeding and increased pneumococcal density should be interpreted with caution. A study comparing the nasopharyngeal microbiome of exclusively breast-fed infants with formula-fed infants found differences in bacterial community composition at six weeks of age that resolved by six months.[40] The generation R study examining risk factors for pneumococcal carriage in Dutch infants found no association between pneumococcal carriage and duration of breast-feeding or exclusive breast-feeding at 1.5, 6 or 14 months of age.[11] Only 20% of participants in the older age group reported current breastfeeding, and it is possible that there may be underlying socioeconomic and other differences between the breastfeeding group and those in the same age group who were no longer breastfed that were not captured therefore not included in the adjusted analysis. Alternatively, it is possible that breastfeeding may negatively affect bacterial species such as Staphylococcus aureus that are competitors of pneumococcus, therefore facilitating pneumococcal growth, although this is speculative.[41]

Viral testing was not conducted in our study as we recruited generally healthy, afebrile children from the community. However, > 60% of participants aged 12–23 months had URTI symptoms. Several studies have identified positive associations between respiratory viruses and pneumococcal carriage and density in the nasopharynx. In children attending day-care in Portugal, pneumococcal density was significantly higher in children who tested positive for a respiratory virus.[14] In a UK clinical trial, children who received the live attenuated influenza vaccine had significantly higher pneumococcal density compared with controls when assessed 28 days after vaccination.[42] In rural children in Peru, respiratory virus detection was positively associated with pneumococcal density.[43] A surveillance study on influenza-like illness in households in Vientiane, Lao PDR found that pneumococcus, influenza virus, and parainfluenza virus were the most common pathogens detected in children aged 0–4 years, and that co-detection of pneumococcus and respiratory viruses was common.[44] Combined, these findings suggest that children in our study with high pneumococcal density and URTI symptoms might have been co-infected with a respiratory virus. However, asymptomatic viral co-infection may also influence pneumococcal density: a study in American children found that pneumococcal densities were higher when a respiratory virus was detected, even in the absence of URTI symptoms.[15]

We did not evaluate HIV infection status or malnutrition, factors that may influence pneumococcal carriage, in our study. Lao PDR has a low prevalence of HIV (estimated at 0.2% of 18 to 49 year olds in 2013) and therefore we assume similar low prevalence in our participants.[45] Childhood malnutrition is a major public health issue in Lao PDR. In 2015, the prevalence of stunting, which is an indicator of chronic malnutrition, in children under five was 36%.[23] Malnutrition was likely common among the study population, and has been identified as a risk factor for pneumococcal carriage in other studies.[46, 47]

In summary, we have identified host and socio-economic risk factors for pneumococcal carriage and density in infants and young children in Lao PDR. Our findings highlight links between URTI infections, pneumococcal carriage, and pneumococcal density, and also identify children living in rural areas as having increased risk of pneumococcal carriage.

We thank study participants and their families, and the University of Health Sciences project staff for enrolment and sample collection. We thank the Lao-Oxford-Mahosot Hospital Wellcome Trust Research Unit for sample processing, and the pneumococcal microbiology lab at the Murdoch Children’s Research Institute and the Bacterial Microarray Group at St George's, University of London for microbiological analysis.

Factors associated with pneumococcal carriage and density in infants and young children in Laos PDR

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Your article has been carefully reviewed by three experts including two physician scientists, one in Pediatrician and one in infectious diseases. They have provided both instructive suggestions and constructive opinions for you to revise this manuscript in a timely manner

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors analyzed the relationship between host factors and pneumococcal carriage density in the young children in the Lao People's Democratic Republic. They provide evidence that some host factors, including living environments, areas, socio-economic factors and URTI infections, would influence the pneumococcal carriage and density in young children. These data provide some information on pneumococcal carriage and density in a high pneumococcal disease burden in Lao, but some important information should be provided more clearly. The details of my concerns are listed as follows.

1. To quantify the bacterial density, the authors use the unit of “genome equivalents per ml (GE/ml)” to represent the bacterial density in this study, but little information about the unit of GE/ml was obtained from this manuscript. The authors should describe how to detect the “genome equivalents per ml” in more detail and tell us what the volume unit represent. They should also provide evidence or reference that the data of “genome equivalents per ml” can represent the absolute number of pneumococci isolated from the nasopharyngeal swab samples. The distribution of bacteria number isolated form the clinical samples are always wide. The result of the pneumococcal density showed in this study is only arranged from 5.49 to 6.00 (log10 GE/ml), which implied the limitation of the quantification assay. Therefore, more detailed description and evidence should be provided to support the presenting data.

2. PVC vaccination will significantly reduce the carriage of PCV13 serotypes (Table 4). Therefore, the carriage of pneumococci or PCV13 serotypes in the group of pre-vaccination should be specifically analyzed.

3. Some results described in the abstract, in the text and showed in the able are not consistent, such as the association of living in a household with two or more children under the age of five years (infants aged 5 - 8 weeks) and pneumococcal carriage. Please check that carefully.

Reviewer #2: The authors identified risk factors for pneumococcal carriage and density using data from cross-sectional pneumococcal carriage surveys conducted in the Lao People's Democratic Republic before and after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13). The authors found that in the age group of 5-8 weeks, having and two or more children under five in the 233 household and low family income were risk factors for pneumococcal carriage. Children aged 12 - 23 months, URTI symptoms, two or more children under five years old in the household, and rural residence were associated with pneumococcal carriage. URTI symptoms, current breastfeeding, rural residence, and delivery by Caesarean section were associated with higher mean pneumococcal density in pneumococcal carriers.

I have some major concerns.

1. The authors have published the other article using the same cohort. (Vaccine. 404 2019;37(2):296-305.) The author should clearly state the difference between the two studies.

2. The pneumococcal density was measured using real-time PCR according to the reference (J Clin Microbiol. 2007;45(8):2460-6.). However, in the initial study by Carvalho, the authors used the real-time PCR to detect rather than quantify the pneumococcus. Several studies are using the same method to quantify the pneumococcus. However, there are augment that measuring the pneumococcus DNA alone is likely to be confounded by variable production and sampling of nasopharyngeal mucous. (Sci Rep. 2018 Jul 23;8(1):11030.). I am wondering in the present study whether the pneumococcal density adjusted for the concentration of human DNA present? If "no", the authors might consider to debate why the adjustment is not necessary.

There also some additional comments.

1. If the data can be delinked, what is the ethical concerns that the data can't be publicly available?

2. Page 2 Line 30. The variable low family income seems statistically not significant.

3. Page 4 Line 73-75. The statement is statistically not significant.

4. Study design and participant section. In what healthcare setting were those participant enrolled? Were the participant of both age group enrolled in the same healthcare setting?

5. page 6, Line 107-110. The author might describe more detail about the method of the realtime PCR. What is used as a standard curve? How the density calculate? What is the detection limits? Did the experiment include positive control or negative control of the swab? Or did the Real-time PCR included positive or negative control?

6. Page 7, Line 132. Is there a preset cutoff for the statistical significance?

7. Page 7, Line 144. According to table 1, there are 507 participants of the age group 12-23 months in the two years post-PCV 13 . Why the denominator is 497 in Line 144 ?

8. Table 1. why the denominator varies in different rows?

9. if the participant of both age group enrolled in the same healthcare setting, it is weird that there are large differences of the distribution of the rural residence and the URTI symptoms.

10. Page 8, Line 155-158. Why are the denominators different? 999 or 995, and 1009 or 1005?

11. Page 9, Line 164-165. The statement is statistically not significant.

13. Page 20, Line 281-282. The post PCV13 carriage survey showed increased pneumococcal density. Except the reference 16 (from the same study cohort as the present study), are there supportive references? or are there possible explanations? The authors might discuss the finding in detail.

Reviewer #3: This study examined the prevalence and density of nasopharyngeal pneumococcal carriage in children of the Lao People's Democratic Republic before and after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13).

In infants aged 5 - 8 weeks, living in a household with two or more children under the age of five years (aOR 1.97; 95% CI 1.39 - 2.79) and low family income (aOR 1.64; 95% CI 0.99 - 2.72) were positively associated with pneumococcal carriage. For children aged 12 - 23 months, upper respiratory tract infection (URTI) symptoms, two or more children under five in the household, and rural residence were positively associated with pneumococcal carriage. PCV13 vaccination was negatively associated with carriage of PCV13 serotypes. URTI symptoms, current breastfeeding, rural residence, and delivery by Caesarean section were associated with higher mean pneumococcal density in pneumococcal carriers.

Overall, this study provides new data on nasopharyngeal carriage in a developing country in South East Asia. Although the methodology is not novel, this study provides new data on pneumococcal burden setting in southeast Asia.

One point needs more elaboration. In this study, current breastfeeding was associated with higher mean pneumococcal density in pneumococcal carriers. This is in contrast to most previous examples about the effect of breast feeding on microbial infection of young children. Although some explanations were raised in the Discussion, more in-depth discussion on the biological basis should be considered.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

The authors thank the reviewers for the time that they put into reviewing our manuscript and for their thoughtful comments and feedback. Line numbers included in our responses refer to the revised version of the manuscript.

Review Comments to the Author

Reviewer #1: In this manuscript, the authors analyzed the relationship between host factors and pneumococcal carriage density in the young children in the Lao People's Democratic Republic. They provide evidence that some host factors, including living environments, areas, socio-economic factors and URTI infections, would influence the pneumococcal carriage and density in young children. These data provide some information on pneumococcal carriage and density in a high pneumococcal disease burden in Lao, but some important information should be provided more clearly. The details of my concerns are listed as follows.

1. To quantify the bacterial density, the authors use the unit of “genome equivalents per ml (GE/ml)” to represent the bacterial density in this study, but little information about the unit of GE/ml was obtained from this manuscript. The authors should describe how to detect the “genome equivalents per ml” in more detail and tell us what the volume unit represent. They should also provide evidence or reference that the data of “genome equivalents per ml” can represent the absolute number of pneumococci isolated from the nasopharyngeal swab samples. The distribution of bacteria number isolated form the clinical samples are always wide. The result of the pneumococcal density showed in this study is only arranged from 5.49 to 6.00 (log10 GE/ml), which implied the limitation of the quantification assay. Therefore, more detailed description and evidence should be provided to support the presenting data.

Authors’ response:

We have provided added information on the definition of GE/ml and how it is calculated, as well as more details on the lytA qPCR assay as suggested by Reviewer #2 (comment 5) to the methods section in lines 112 - 119. Consistent with other studies, we also observed a wide distribution of pneumococcal density, ranging from 2.9 to 8.2 log10 GE/ml. We have added these data to the results section in lines 179 - 181, and noted the wide distribution in the discussion (lines 301 - 302). Note that only mean densities are presented in Table 5 in the interest of space and do not represent the full range of densities observed.

2. PVC vaccination will significantly reduce the carriage of PCV13 serotypes (Table 4). Therefore, the carriage of pneumococci or PCV13 serotypes in the group of pre-vaccination should be specifically analyzed.

Authors’ response:

We conducted an additional analysis as suggested to examine carriage of PCV13 serotypes in 12-23 month old children specifically in the pre-PCV survey. Results were consistent with the original results (from both surveys combined) shown in Table 4, with rural residence, upper respiratory tract symptoms, and living in a household with two or more children under the age of five years found to be associated with PCV13 serotype carriage. The results of this additional analysis have been included in the results section in lines 219 - 226.

3. Some results described in the abstract, in the text and showed in the able are not consistent, such as the association of living in a household with two or more children under the age of five years (infants aged 5 - 8 weeks) and pneumococcal carriage. Please check that carefully.

Author’s response:

Thank you for that observation. We have checked all of the data and fixed the error identified in the results text as well as a couple of other typos identified.

Reviewer #2: The authors identified risk factors for pneumococcal carriage and density using data from cross-sectional pneumococcal carriage surveys conducted in the Lao People's Democratic Republic before and after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13). The authors found that in the age group of 5-8 weeks, having and two or more children under five in the 233 household and low family income were risk factors for pneumococcal carriage. Children aged 12 - 23 months, URTI symptoms, two or more children under five years old in the household, and rural residence were associated with pneumococcal carriage. URTI symptoms, current breastfeeding, rural residence, and delivery by Caesarean section were associated with higher mean pneumococcal density in pneumococcal carriers.

I have some major concerns.

1. The authors have published the other article using the same cohort. (Vaccine. 404 2019;37(2):296-305.) The author should clearly state the difference between the two studies.

Authors’ response:

The objective of the original study published in Vaccine was to evaluate the impact of PCV13 introduction on pneumococcal carriage and circulating serotypes. Specifically, we compared carriage rates pre- and 2 years post PCV13 introduction. The objective of this current manuscript was to identify demographic and household risk factors for pneumococcal carriage and pneumococcal carriage density. We have added the following information to the introduction (lines 75 - 77) to clearly highlight the differences between these two studies:

“Here, we describe a secondary analysis conducted using data from these cross-sectional surveys to identify demographic and household factors associated with carriage of pneumococci overall, PCV13 serotype carriage, and pneumococcal carriage density.”

2. The pneumococcal density was measured using real-time PCR according to the reference (J Clin Microbiol. 2007;45(8):2460-6.). However, in the initial study by Carvalho, the authors used the real-time PCR to detect rather than quantify the pneumococcus. Several studies are using the same method to quantify the pneumococcus. However, there are augment that measuring the pneumococcus DNA alone is likely to be confounded by variable production and sampling of nasopharyngeal mucous. (Sci Rep. 2018 Jul 23;8(1):11030.). I am wondering in the present study whether the pneumococcal density adjusted for the concentration of human DNA present? If "no", the authors might consider to debate why the adjustment is not necessary.

Authors’ response:

We did not adjust for human DNA concentration in this study. Our laboratory has considered whether this approach would be useful for pneumococcal carriage studies, and upon careful consideration we determined that it would not be of sufficient benefit to implement. As human DNA greatly exceeds pneumococcal DNA in a swab sample, the alu qPCR described in the 2018 Sci Reports paper requires a 1:1000 dilution of template DNA. This necessitates additional manual handling of specimens, which is a risk for contamination, particularly cross-contamination between samples. Running a second qPCR assay would also incur additional labor and consumables costs. Notably, the authors of this paper report that “the adjustment made little difference to the results, suggesting that the nasopharyngeal flocked swab is efficient at collecting a standard volume of secretion from the posterior nasopharyngeal mucosa.” As the adjustment made little difference to the results, while increasing the laboratory workload and risk of contamination, we chose not to implement this procedure. To help ensure consistent sampling of the nasopharynx, swab collection was conducted by trained study personnel, performed using flocked swabs, and swab collection, transport, and storage were conducted in accordance with WHO recommendations (Vaccine. 2013;32(1):165-79).

There also some additional comments.

1. If the data can be delinked, what is the ethical concerns that the data can't be publicly available?

Authors’ response:

The ethical concerns preventing making data publicly available are not due to personally identifying information, which as noted, can be delinked. Rather, the issue is that the protocol that was approved by ethics committees in Lao PDR and Australia specified the purpose of the study and what data would be used for, and this information was also conveyed in the informed consent process. Therefore, reasonable requests for data would need to include details on how the data would be used and will be subject to approval by the Lao PDR Ministry of Health National Ethics Committee for Health Research, which is the overseeing ethics committee for this study. Additionally, this process is mindful of potential sensitivities regarding data from ethnic minorities.

2. Page 2 Line 30. The variable low family income seems statistically not significant.

Authors’ response:

In keeping with the recent movement away from using p < 0.05 as a strict cutoff for statistical significance (see Wasserstein and Lazar. The ASA Statement on p-Values: Context, Process, and Purpose. The American Statistician 2016 70(2): 129-133; Harrington et al. New Guidelines for Statistical Reporting in the Journal. N Engl J Med. 2019 Jul 18;381(3):285-286.) throughout the manuscript we have presented p values and 95% confidence intervals (CI) and taken these measures as well as the odds ratio into account when interpreting results, and avoided describing our results as ‘statistically significant’. Our interpretation is that the adjusted odds ratio of 1.64 (95% CI 1.39 – 2.79) provides some evidence of a positive association, as described in the results (lines 185 - 186). In the abstract this description has been shortened however the reader can interpret the data using the 95% CI.

3. Page 4 Line 73-75. The statement is statistically not significant.

Authors’ response:

These results are described as showing “some evidence of indirect effects” and not statistically significant in the text, with an explanation provided above.

4. Study design and participant section. In what healthcare setting were those participant enrolled? Were the participant of both age group enrolled in the same healthcare setting?

Authors’ response:

Participants of both age groups were primarily enrolled during routine clinic visits, however some rural participants were enrolled via household visits. We have added this information to the relevant section of the methods (lines 84 - 85).

5. page 6, Line 107-110. The author might describe more detail about the method of the realtime PCR. What is used as a standard curve? How the density calculate? What is the detection limits? Did the experiment include positive control or negative control of the swab? Or did the Real-time PCR included positive or negative control?

Authors’ response: In response to these questions and those of Reviewer #1 (comment 2), we have added more information about the qPCR assay, standard curve, controls, and how density is calculated to the methods section (lines 112 – 119).

6. Page 7, Line 132. Is there a preset cutoff for the statistical significance?

Authors’ response: As described in the response to comment 2, we did not use a preset cutoff for statistical significance.

7. Page 7, Line 144. According to table 1, there are 507 participants of the age group 12-23 months in the two years post-PCV 13 . Why the denominator is 497 in Line 144 ?

Authors’ response:

Information on vaccine history was unable to be verified from written records for 10 participants. We have added this information to the results in lines 156 – 157.

Table 1. why the denominator varies in different rows?

Authors’ response:

Some data were missing for particular variables, therefore these participants were excluded when percentages were calculated for that variable. We have edited the footnote to explain this.

9. if the participant of both age group enrolled in the same healthcare setting, it is weird that there are large differences of the distribution of the rural residence and the URTI symptoms.

Authors’ response:

We agree that this is a noticeable difference. As for the low proportion of infants aged 5 – 8 weeks enrolled in rural areas, we recruited in one province over a relatively short time frame, and due to a lower populations size and number of births, the number of potentially eligible participants in this age group was smaller than those residing in urban areas. We have mentioned the low proportion of 5 – 8 week olds from rural areas in the discussion (lines 275 - 277).

In terms of URTI symptoms, results are consistent with studies conducted by us and others showing that URTI symptoms are more common in older infants and toddlers than very young infants (Kusel et al. Pediatr Infect Dis J. 2006 Aug;25(8):680-6; Dunne et al. Lancet Glob Health. 2018 Dec;6(12):e1375-e1385.) The most likely explanation for this is the lower exposure of very young infants to respiratory viruses, as they are more likely to be kept indoors and typically do not attend school or day care. Traditionally in Lao PDR, for the first month of an infant’s life, both mother and baby stay within the home, with limited visitors. These practices would reduce exposure to URTIs in the young infant age group.

10. Page 8, Line 155-158. Why are the denominators different? 999 or 995, and 1009 or 1005?

Authors’ response:

The denominators are different as specimens that were not available for serotyping were excluded, therefore the denominators differed by group depending on the number of available samples. We have added a sentence in the methods noting that when serotyping results were not available due to technical issues, that sample was excluded from serotype-specific analyses (lines 142 - 143).

11. Page 9, Line 164-165. The statement is statistically not significant.

Authors’ response:

The adjusted odds ratio of 1.64 (95% CI 1.39 – 2.79) is described as ‘some evidence for a positive association’ in line with our interpretation of these data, as explained in response to comment 2.

We have added the pseudo R2 or adjusted R2 values as appropriate to table footnotes.

13. Page 20, Line 281-282. The post PCV13 carriage survey showed increased pneumococcal density. Except the reference 16 (from the same study cohort as the present study), are there supportive references? or are there possible explanations? The authors might discuss the finding in detail.

Authors’ response:

Published data on the effects of PCV and pneumococcal density have shown varying results. As suggested, we have expanded the discussion and cited additional references (lines 309 - 318).

Reviewer #3: This study examined the prevalence and density of nasopharyngeal pneumococcal carriage in children of the Lao People's Democratic Republic before and after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13).

In infants aged 5 - 8 weeks, living in a household with two or more children under the age of five years (aOR 1.97; 95% CI 1.39 - 2.79) and low family income (aOR 1.64; 95% CI 0.99 - 2.72) were positively associated with pneumococcal carriage. For children aged 12 - 23 months, upper respiratory tract infection (URTI) symptoms, two or more children under five in the household, and rural residence were positively associated with pneumococcal carriage. PCV13 vaccination was negatively associated with carriage of PCV13 serotypes. URTI symptoms, current breastfeeding, rural residence, and delivery by Caesarean section were associated with higher mean pneumococcal density in pneumococcal carriers.

Overall, this study provides new data on nasopharyngeal carriage in a developing country in South East Asia. Although the methodology is not novel, this study provides new data on pneumococcal burden setting in southeast Asia.

One point needs more elaboration. In this study, current breastfeeding was associated with higher mean pneumococcal density in pneumococcal carriers. This is in contrast to most previous examples about the effect of breast feeding on microbial infection of young children. Although some explanations were raised in the Discussion, more in-depth discussion on the biological basis should be considered.

Authors’ response:

We have added some additional possible explanations of this result to the discussion as suggested (lines 335 - 341). These include possible confounding- there may something different about infants breast fed for longer that is also associated with higher pneumococcal density. One plausible biological explanation could due to indirect effects- breast feeding has an inhibitory effect on a bacterium such as S. aureus which is a competitor of pneumococcus.

________________________________________

Attachment

Submitted filename: response to reviewers.docx

10.1371/journal.pone.0224392.r003Decision Letter 1ChiaJean-SanAcademic Editor2019Jean-San ChiaThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Submission Version1

17 Sep 2019

PONE-D-19-19269R1

Factors associated with pneumococcal carriage and density in infants and young children in Laos PDR

PLOS ONE

Dear Dr Dunne,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Kind regards,

Jean-San Chia

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Please address the concerns of reviewer one. [The authors have addressed all my concerns, but I still have a minor suggestion. The range of each data of bacterial density can be provided to make the presenting data more clear.]

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed all my concerns, but I still have a minor suggestion. The range of each data of bacterial density can be provided to make the presenting data more clear.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

The authors thank the editor and reviewers for their consideration of our revised manuscript and the feedback provided. Please find a response to reviewer comments below. Line numbers refer to the clean, final version of the manuscript.

Review Comments to the Author

Reviewer #1: The authors have addressed all my concerns, but I still have a minor suggestion. The range of each data of bacterial density can be provided to make the presenting data more clear.

Authors’ response:

As suggested, we have added ranges of bacterial density data in the results text (lines 180 – 181) and to Table 5.

Attachment

Submitted filename: response to reviewers.docx

10.1371/journal.pone.0224392.r005Decision Letter 2ChiaJean-SanAcademic Editor2019Jean-San ChiaThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Submission Version2

14 Oct 2019

Factors associated with pneumococcal carriage and density in infants and young children in Laos

PONE-D-19-19269R2

Dear Dr. Dunne,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Jean-San Chia

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

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6. Review Comments to the Author

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Reviewer #1: The authors have addressed all my concerns. It has been a well-written manuscript. I have no other comments.

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Reviewer #1: No

10.1371/journal.pone.0224392.r006Acceptance letterChiaJean-SanAcademic Editor2019Jean-San ChiaThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

21 Oct 2019

PONE-D-19-19269R2

Factors associated with pneumococcal carriage and density in infants and young children in Laos PDR

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